Preparation of specific antigen coctoimmunogen



f Patented Nov. 24, 1931 UNITED STATE-s:

RYUZET TOME-Arm.

or Osaka; tartan PREPARATION or srnorrm nn rmnn oooronv'r m-onoenw V rThe present invention relates toa prep r tion of specific antigens whichare free or nearly free from secondary reactions. The} method ofpreparing. the antigens is as 01 '5' lows :Either the pure culture ofthe bact'er antigen, which is employed for the prophy-k lactic ortherapeutic purpose. The object, of the preparation is toovercomevariousdefects in vaccines previously known and "to obtain a. vaccinewhich may serve for the above mentionedpurposes not onlybyinjection, butby being. administered peross For convenience, the present invention-Ymay be explained by d-ividing; it into three parts as follows: 1 i pArticle L-The principlesiof the, present invention are based upon thefollowing-facts; which have been established by experiment andscientific investigation; In the pure cul ture of any bacterium or inthe hitherto known vaccines, there is :a serological sub stance whichinterrupts the mutual action; of the antigen with the antiserum Thissubstance has been proved to-be destroyed by exposing I the culture of thevaccine to: Roentgen rays, ultra violet rays 'orboiling heat at100 C.The inventor has named this substance Impedin. The time re quired forthe complete destruction ofthe-j Impedin differs with the dlifiere'ntspecies of the bacteriaand" is. decided by quantitative determinationofthema'xi'mum production of sedimentation by precipitation reactionswith the antigen and the corresponding antiserum. The quantitativedetermination a is conveniently made by means ot "the precipitonieter,

which the present inventor hasinvented The antigen .isfree'd from: thebacterial bodies and the freed antigen is diSSCLVGdJiiIltQ :the.

1922; SeriaL Idol svs'naw liquid by heating. The immunizing-props. ertyis practically absent in: theresidue ot the boiled bacterial bodiesafterthe extraction of the a soluble I antigen, but is present in the,filtrate of the boiled bacterial culture,v

The number of the bacteriali bodies: doesn otj constitute any ground onwhich theefiicacy off the antigen can be estimated; H OWeVer, with. thefiltrates of the boiled emulsiom the efficacy ofimmunizati on beestimatediby the quantity of the sedimentation is pro; ducedlvby theprecipitating reaction. The amount ofthelprecipitateniay giveianiildicati'on ofthe strength ofthe antigemtr other words; the effect-sot theprecipitinogen; and the antigen-- are proportional o'rlp'arallel"toeaclivother; I 1 r Vaccines heretofore known have varieusobjectionable secondary 'efi'ects, for. example, yr'exi'a reacts toproducea local swellingfandf pain] at; the laced, inj e-etion,fetelsrbutjthe boiled"antigen treed from theresidue r micro-organlsmsdoes. IlOtglVejlIlX notice able secondary effect;-

The method of; are inventionv will: be: ex plained more minutely.thepreparationofe ref example, typhoid" antigen. fAjtwent'yy tour-twentyeight hour old culture ofBdbil-T lies [fig ollows is cultivated on asolid mediuin of common agar'and is suspended ;in physil l ological saltsolution inthe proportion 03512: mglto 1 cQc. The suspension is, for.instance;

divided into 'five' squat peas, and the parts i I-IV are'heated at 100C. forQ O, 30, 4'0 and" 60fminutes respectively; and whiletlie part. V"

remainsunheated", each part is then sterilely filtered through 'a'porce'lain iilter. Each fil t'rate isthe'n mixed" with *the samequantity of typhoid-'imniune serum, and the quantity of-theprecipitation isestiina-t'ed by means 'of the precipitoineter'. If the.mixture of the immune serum andthe sterile filtrateof the suspensionthat had been heateddio'r one-hart hour should give the maximum quantityoi the precipitate, the limped-inproduced bythe hour. andthe'sedimentation' removed either by filtration through the porcelainfilter or by other routine methods. The clear filtrate is hermeticallysealed and thus the preparation of the antigen is completed. I

It has been found that either Roentgen rays or ultra violet rays giveabout the same results as the application of heat infthe de struction ofImpedin, but for'practical purposes the boiling of the aqueoussuspension is the simplest method of preparation.

' The'immunization value of'the boiled an tigen, as it is prepared afterthe above mentioned method, is not expressed by the number ofthebacteria as is made in the hitherto known vaccines, but by the totalquantity of the precipitate produced in a mixture of equal volumes ofthe antigen and of standard immune serum. .7 I p Article .l[.This methodfacilitates the preparation of the antigen with a certain species of thepathogenic micro-organisms with Which the coctoimmunogen cannot bemethod of the'preparation is explained by tion and filtration. The soobtained trans parent fluid is hermetically sealed in glass prepared bythe method statedin the Article I, or with a species of the pathogenicmicroorganisms withwhich the pure culture has not been able to beproduced. Or in other words,the present method described inthis Yarticle has thecharacteristic to facilitatethe' preparation'of theantigen,'by exposing to the boiling heat at 100 (1., Roentgen rays orultraviolet rays the'emulsion of the animal tissues that was obtainedfromthe animals infected with the pure culture of the pathogenicmicro-organisms or the naturally infected human or animal pathologicaltis' sues, from which the pathogenic micro-or-.

ganisms can not yet be cultivated. The

the following two or'three'instances. q p

' The visceraof the animal that has been infect ed by typhus fever, e.g., the spleeln'has been proved to contain'a great 'deal" of the 7antigen. Therefore, a physiological saline emulsion is prepared with ithe infected spleen, and heated at 100 C. for one/half hour and thenentirely freed from any in soluble substances byjmeans or"centrifugabottles. This is called the specific boiled antigen,.ornon-bacterialvaccine, or coctoiinmunogen of typhus fever. This completesthe entire process of the preparation. 7 Another instance :Aphysiological saline emulsion is prepared with the lymph collected fromthe 7-8 day old ripe vacciniaof either naturally orartificially infectedcow inproportion of 1.0 g. i 5.0 c. c. 7 The emul sion is then boiled inthe water bath for one half hour to one hour and centrifugalized. Thesupernatant fluid is the specific antigen,;which is filled in glasstubes and I hermetically sealed. This completes the preparation of thevaccinia cocto antigen. 7

Thus the-present invention may be applied to the preparation of antigenswith various invisible pathogenic microorganisms such tract of theviscera'infected with the bacteria that can be purely cultivated ontheartificial medium has a stronger 'specific'antigenic property thanthevaccine consisting ofthe i bacterial emulsion. Therefore, a strongantlgen may be obtained even w th the genie micro-organisms that areliable to be path0- 5 V attenuated in consequence ofthe cultivation Iforseveral generations by boiling theemul v sion of thevisceraartificially infected with the pure culture of such attenuated germ.

Article ll[. All the hitherto known vaccines, which are used either'forprophylactic or therapeutic purposes, consist of nothing but asuspension of antigenic bacteria, and are employed exclusively byinjection. The injection is, however, not absolutely necessary for theadministration of antigens,

because all the infectious diseases, which are related with thedigestive tracts such as cholera, 1

typhoid fever, dysentery, etc., maybe prophylactically controlledby thelocal immuni .zation that is bestowed on the tracts by theadministration of the antigen per os.

In order to employ the suspension of'the i antigenic bacteriaas all ofthe hitherto known vaccines are formed, for the internal administration,there are met with not only various inconveniences in their.transportation and uses, but also the deteriorationof the vaccinesthemselves by being kept'for 'a long time. These facts make them unfit.for their preservation. The present article deals with the method ofpreparing the desiccated antigen, which has been originated by thepresent inventor.

Theminute description of the. method is given below: For instance, inthe preparation of the desiccated cholera antigen, the pure' culture ofVibrz'o cholera; is exposed to the boiling heat at 100 C.,'Roentgen raysor ultraviolet rays, just after the same manner as. has been describedin Articles Ijand II. The specific antigen of Vibrio choleraa,consisting of the residuefof the bacterial bodies,

is mixed with an appropriate ingredient such as magnesia usta inproportion ofa certain H quantity, of the boiled specific antigen justequal to the antigenic strength as 0.2 milln.

gram of the bacteria per 0.1 gram oftheimixture. The mixture is thendesiccated into iao powder, and it is bottled in the form of powtissuebeing of a kind that responds to said der, pills or tablets, each pieceof the latter two being made to contain 0.1 gram of the antigen. Thiscompletes the preparation of the desiccated antigen;

This invention is based on the principle which the inventor advocatesthat the purpose of the prophylactic treatment of an infectious diseaseof the digestive tracts is most rationally to be eflected by bestowingimmunization on the canal itself instead of inj ecting the vaccinehypodermically as hitherto has been used in practice. As there is noneed of apprehension as to the by-efiects such as pyreXia, localswelling or pain, etc., the antigen for the purpose of the intestinaladministration per os. can be practically made int-o powder immediatelyafter the bacterial emulsion has been exposed to heat, Roentgen rays orultraviolet rays without clearing of the bacterial residues, as has beenstated in the preceding paragraphs. I f

The desiccated antigens of Bacillus typhosus, Uolz' comm'cmz's paratgphosus, Bacillus rlg sem erz'ac, etc. may be obtained by applying thesame method as stated above, only the difference being that Vibrooholewc is replaced by any one of these bacteria.

Having now described my invention, what I claim is 1. The method ofpreparing an antigen subjecting such suspension to the action of anagent capable of preventing such inherent tendency, and therebyfacilitate the action of antibodies, complements and leucocytes, thebacterial substance being of a kind that responds to said action of saidagent.

2. The method of preparing an antigen from a bacterial substance havinginherently a tendency to impede the action oftne antigen which includesthe acts of providing a culture of bacteria that normally contains asubstance inherently tending to impede the action of the antigen andboiling said culture under temperature and time conditions until saidimpeding substance is destroyed and thereby to facilitate the action ofantibodies,

complements and leucocytes, the bacteria of i the culture being of akind that responds to such treatment.

3. The method of preparing an antigen from a bacterial substance havinginherently a tendency to impede the action of the antigen which includesthe acts of providing a physiological saline emulsion ofanimalpathological tissue, which contains a substance that tends toimpede the action of the antigen and subjecting said emulsion to theaction of an agent capable of destroying said impeding substance andthereby facilitate the action of antibodies, complements and leucocytes,said treatment.

4. A method as in claim a in which the boil- DR. R. TORIKATA.

